Genevieve Evin

Increased expression of the amyloid precursor β-secretase in Alzheimer's disease

β‐Secretase cleavage represents the first step in the generation of Aβ polypeptides and initiates the amyloid cascade that leads to neurodegeneration in Alzheimers disease. By comparative Western blot analysis, we show a 2.7‐fold increase in protein expression of the β‐secretase enzyme BACE in the brain cortex of Alzheimers disease patients as compared to age‐matched controls. Similarly, we found the levels of the amyloid precursor protein C‐terminal fragment produced by β‐secretase to be increased by nearly twofold in Alzheimers disease cortex.

Degradation of the Alzheimer Disease Amyloid β-Peptide by Metal-dependent Up-regulation of Metalloprotease Activity

Biometals play an important role in Alzheimer disease, and recent reports have described the development of potential therapeutic agents based on modulation of metal bioavailability. The metal ligand clioquinol (CQ) has shown promising results in animal models and small phase clinical trials; however, the actual mode of action in vivo has not been determined. We now report a novel effect of CQ on amyloid β-peptide (Aβ) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu2+ or Zn2+ resulted in an ∼85–90% reduction of secreted Aβ-(1–40) and Aβ-(1–42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted Aβ was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu2+. MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu2+ also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of Aβ-(1–40). Our findings identify an alternative mechanism of action for CQ in the reduction of Aβ deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients.

Proteolytic Processing of the Alzheimer’s Disease Amyloid Precursor Protein within Its Cytoplasmic Domain by Caspase-like Proteases

Alzheimer’s disease is characterized by neurodegeneration and deposition of βA4, a peptide that is proteolytically released from the amyloid precursor protein (APP). Missense mutations in the genes coding for APP and for the polytopic membrane proteins presenilin (PS) 1 and PS2 have been linked to familial forms of early-onset Alzheimer’s disease. Overexpression of presenilins, especially that of PS2, induces increased susceptibility for apoptosis that is even more pronounced in cells expressing presenilin mutants. Additionally, presenilins themselves are targets for activated caspases in apoptotic cells. When we analyzed APP in COS-7 cells overexpressing PS2, we observed proteolytic processing close to the APP carboxyl terminus. Proteolytic conversion was increased in the presence of PS2-I, which encodes one of the known PS2 pathogenic mutations. The same proteolytic processing occurred in cells treated with chemical inducers of apoptosis, suggesting a participation of activated caspases in the carboxyl-terminal truncation of APP. This was confirmed by showing that specific caspase inhibitors blocked the apoptotic conversion of APP. Sequence analysis of the APP cytosolic domain revealed a consensus motif for group III caspases ((IVL)ExD). Mutation of the corresponding Asp664 residue abolished cleavage, thereby identifying APP as a target molecule for caspase-like proteases in the pathways of programmed cellular death.

Biogenesis and metabolism of Alzheimer’s disease Aβ amyloid peptides

Biochemical and genetic evidence indicates the balance of biogenesis/clearance of Abeta amyloid peptides is altered in Alzheimers disease. Abeta is derived, by two sequential cleavages, from the receptor-like amyloid precursor protein (APP). The proteases involved are beta-secretase, identified as the novel aspartyl protease BACE, and gamma-secretase, a multimeric complex containing the presenilins (PS). Gamma-secretase can release either Abeta40 or the more aggregating and cytotoxic Abeta42. Secreted Abeta peptides become either degraded by the metalloproteases insulin-degrading enzyme (IDE) and neprilysin or metabolized through receptor uptake mediated by apolipoprotein E. Therapeutic approaches based on secretase inhibition or amyloid clearance are currently under development.

Proteolytic processing of the Alzheimer's disease amyloid precursor protein in brain and platelets

Proteolytic processing of the amyloid precursor protein by β ‐and γ‐secretases results in the production of Alzheimers disease (AD) Aβ amyloid peptides. Modulation of secretase activity is being investigated as a potential therapeutic approach. Recent studies with human brain have revealed that the β‐secretase protein, BACE, is increased in cortex of AD patients. Analysis of βCTF (or C99), the amyloid precursor protein (APP) product of BACE cleavage that is the direct precursor to Aβ, shows it is also elevated in AD, underlying the importance of β‐secretase cleavage in AD pathogenesis. The C‐terminal product of γ‐secretase cleavage of APP, ϵCTF (or AICD), is enriched in human brain cortical nuclear fractions, a subcellular distribution appropriate for a putative involvement of APP cytosolic domain in signal transduction. Analysis of AD cortex samples, particularly that of a carrier of a familial APP mutation, suggests that processing of APP transmembrane domain generates an alternative CTF product. All these particularities observed in the AD brain demonstrate that APP processing is altered in AD. The transgenic mouse model Tg2576 seems to be a promising laboratory tool to test potential modulators of Aβ formation. Indeed, C‐terminal products of α‐, β‐, and γ‐secretase cleavage are readily detectable in the brain of these transgenic mice. Finally, the finding of the same secretase products in platelets and neurons make platelets a potentially useful and easily accessible clinical tool to monitor effects of novel therapies based on inhibition of β‐ or γ‐secretase.

An alternative strategy for the radioimmunoassay of angiotensin peptides using amino-terminal-directed antisera: measurement of eight angiotensin peptides in human plasma.

We describe here a method of measuring angiotensin peptides and their carboxy-truncated metabolites in human plasma using N-terminal-directed antisera. Antisera raised against N-acetylated angiotensin (Ang) II and N-acetylated Ang III analogues were used to develop two radioimmunoassays. Extracted plasma samples were acetylated prior to separation of cross-reacting angiotensin peptides by high-performance liquid chromatography (HPLC). Fractions were assayed with both antisera to obtain measurements for eight angiotensin peptides. Angiotensin levels measured in normal males were (fmol/ml plasma, mean +/- s.e.m., n = 14): Ang-(1-7) 1.0 +/- 0.2, Ang II 13.9 +/- 2.0, Ang-(1-9) less than 0.4, Ang I 19.5 +/- 2.4, Ang-(2-7) less than 1.1, Ang III 2.9 +/- 1.0, Ang-(2-9) less than 2.1, Ang-(2-10) 2.4 +/- 0.8. Hypertensive patients receiving angiotensin converting enzyme (ACE) inhibitor therapy (n = 8) had an increase in Ang I to 187.3 +/- 107.2 fmol/ml (P = 0.002), and a reduction in Ang II to 4.8 +/- 1.2 fmol/ml (P less than 0.001). Furthermore, these patients showed a ninefold increase in Ang-(1-7) to 9.7 +/- 4.3 fmol/ml (P less than 0.001), indicating a role for prolylendopeptidase in the metabolism of Ang I in vivo. These N-terminal assays have demonstrated that carboxy-truncated metabolites of Ang I and Ang II make little contribution to plasma angiotensin peptides, except during ACE inhibitor therapy. Furthermore, these antisera allow the measurement of Ang I and Ang II in the same radioimmunoassay of fractions from HPLC, providing a highly reliable estimate of the Ang II:Ang I ratio.

Inhibition of γ-Secretase as a Therapeutic Intervention for Alzheimer’s Disease

Genetic and experimental evidence points to amyloid-β (Aβ) peptide as the culprit in Alzheimer’s disease pathogenesis. This protein fragment abnormally accumulates in the brain cortex and hippocampus of patients with Alzheimer’s disease, and self-aggregates to form toxic oligomers causing neurodegeneration.Aβ is heterogeneous and produced from a precursor protein (amyloid precursor protein [APP]) by two sequential proteolytic cleavages that involve β- and γ-secretases. This latter enzyme represents a potentially attractive drug target since it dictates the solubility of the generated Aβ fragment by creating peptides of various lengths, namely Aβ40 and Aβ42, the longest being the most aggregating. γ-Secretase comprises a molecular complex of four integral membrane proteins — presenilin,nicastrin, APH-1 and PEN-2 — and its molecular mechanism remains under extensive scrutiny. The ratio of Aβ42 over Aβ40 is increased by familial Alzheimer’s disease mutations occurring in the presenilin genes or in APP, near the γ-secretase cleavage site.Potent γ-secretase inhibitors have been identified by screening drug libraries or by designing aspartyl protease transition-state analogues based on the APP substrate cleavage site. Most of these compounds are not specific for γ-secretase cleavage of APP, and equally inhibit the processing of other γ-secretase substrates, such as Notch and a subset of cell-surface receptors and proteins involved in embryonic development, haematopoiesis, cell adhesion and cell/cell contacts. Therefore, current research aims at finding compounds that show selectivity for APP cleavage, and particularly that inhibit the formation of the aggregating form, Aβy42. Compounds that target the substrate docking site rather than the enzyme active site are also being investigated as an alternative strategy. The finding that some NSAID analogues preferentially inhibit the formation of Aβ42 over Aβ40 and do not affect Notch processing has opened a new therapeutic window. The progress in design of selective inhibitors as well as recent results obtained in animal studies prove that γ-secretase remains among the best targets for the therapeutic control of amyloid build-up in Alzheimer’s disease. The full understanding of γ-secretase regulation may yet uncover new therapeutic leads.

APP intracellular domain is increased and soluble Aβ is reduced with diet-induced hypercholesterolemia in a transgenic mouse model of Alzheimer disease

Cholesterol is one of multiple factors, other than familial genetic mutations, that can influence amyloid-beta peptide (Abeta) metabolism and accumulation in Alzheimer disease (AD). The effect of a high-cholesterol diet on amyloid precursor protein (APP) processing in brain has not been thoroughly studied. This study was designed to further investigate the role of cholesterol in the production of Abeta and APP intracellular domain (AICD) in 12-month-old Tg2576 transgenic mice. The mice were maintained on a high-cholesterol diet for 6 weeks. We found that diet-induced hypercholesterolemia increased the APP cytosolic fragment AICD and reduced sAPPalpha in the Tg2576 mice compared to the mice on a control basal diet. In addition, the levels of detergent-extracted Abeta40 were reduced, although no change in guanidine-extracted Abeta levels was observed. Full-length APP, alpha/betaC-terminal fragment (alpha/betaCTF), and beta-secretase (BACE) were not different in the cholesterol-fed mice compared to the control diet-fed mice. This study suggests that a high dietary cholesterol in aged mice may not only influence Abeta metabolism, but also regulate the AICD levels. AICD has a proposed role in signal transduction and apoptosis, hence modulation of AICD production could be an alternative mechanism by which cholesterol contributes to AD pathogenesis.

ALZHEIMER'S DISEASE-ASSOCIATED PRESENILIN 1 IN NEURONAL CELLS : EVIDENCE FOR LOCALIZATION TO THE ENDOPLASMIC RETICULUM-GOLGI INTERMEDIATE COMPARTMENT

The recently identified Alzheimers disease‐associated presenilin 1 and 2 (PS1 and PS2) genes encode two homologous multi membrane‐spanning proteins. Rabbit antibodies to the N‐terminal domain of PS1 detected PS1 in human neuroblastoma SH‐SY5Y wild type and PS1 transfectants (SY5Y‐PS1) as well as in mouse P19, in CHO‐K1 and CHO‐APP770 transfected cells, in rat cerebellar granule and hippocampal neurons, and astrocytes. Immunoblotting detected full‐length protein of 50 kDa, and a major presumptive cleavage product of 30 kDa. The immunofluorescence pattern resembled labeling of the endoplasmic reticulum‐Golgi intermediate compartment (ERGIC) marker protein ERGIC‐53. PS1 distribution showed slight condensation after brefeldin A and more marked condensation after incubation of cells at 16°C, characteristic of the ERGIC compartment. Double labeling showed colocalization of ERGIC‐53 with PS1 in the SY5Y‐PS1 cells. PS1 labeling of SY5Y‐PS1 and P19 cells showed overlap of the cis‐Golgi marker p210 and colocalization with p210 after brefeldin A which causes redistribution of p210 to the ERGIC. Expression of PS1 did not change in level or cellular distribution during development of neurons in culture. Double labeling for the amyloid precursor protein (APP) and PS1 on SY5Y‐PS1 cells and CHO‐APP770 cells showed some overlap under control conditions. These results indicate that PS1 is a resident protein of the ERGIC and could be involved in trafficking of proteins, including APP, between the ER and Golgi compartments. J. Neurosci. Res. 49:719–731, 1997.

p75NTR Regulates Aβ Deposition by Increasing Aβ Production But Inhibiting Aβ Aggregation with Its Extracellular Domain

Accumulation of toxic amyloid-β (Aβ) in the cerebral cortex and hippocampus is a major pathological feature of Alzheimers disease (AD). The neurotrophin receptor p75NTR has been proposed to mediate Aβ-induced neurotoxicity; however, its role in the development of AD remains to be clarified. The p75NTR/ExonIII−/− mice and APPSwe/PS1dE9 mice were crossed to generate transgenic AD mice with deletion of p75NTR gene. In APPSwe/PS1dE9 transgenic mice, p75NTR expression was localized in the basal forebrain neurons and degenerative neurites in neocortex, increased with aging, and further activated by Aβ accumulation. Deletion of the p75NTR gene in APPSwe/PS1dE9 mice reduced soluble Aβ levels in the brain and serum, but increased the accumulation of insoluble Aβ and Aβ plaque formation. There was no change in the levels of amyloid precursor protein (APP) and its proteolytic derivatives, or α-, β-, and γ-secretase activities, or in levels of BACE1, neprilysin (NEP), and insulin-degrading enzyme (IDE) proteins. Aβ production by cortical neurons of APPSwe/PS1dE9 mice was reduced by deletion of p75NTR gene in vitro. Recombinant extracellular domain of p75NTR attenuated the oligomerization and fibrillation of synthetic Aβ42 peptide in vitro, and reduced local Aβ plaques after hippocampus injection in vivo. In addition, deletion of p75NTR attenuated microgliosis but increased the microhemorrhage profiles in the brain. The deletion of p75NTR did not significantly change the cognitive function of the mice up to the age of 9 months. Our data suggest that p75NTR plays a critical role in regulating Aβ levels by both increasing Aβ production and attenuating its aggregation, and they caution that a therapeutic intervention simply reducing p75NTR may exacerbate AD pathology.